Synthetic oligonucleotides play a pivotal role in molecular biology research, useful especially for DNA sequencing, DNA amplification, and hybridization. A novel method for the synthesis of oligonucleotides has been described previously by the inventor in U.S. patent applications Ser. Nos. 07/995,791 and 08/100,671 to replace both the obsolete enzymatic methods and the current chemical methods. This method, referred to herein as the "One Pot" method basically involves repeated cycles of extending a primer chain using a nucleotide having a 3'-blocking group, thus forming an extended primer with a blocking group at its 3'-end; inactivation of excess nucleotide; and removal of the 3'-blocking group from the extended primer to prepare the extended primer for the addition of the next nucleotide. The ease with which this method can be automated will foster a new generation of oligonucleotide synthesizers with enormous throughput, increased reliability, lower cost per synthesis, and with environmentally friendly reagents.
One limitation associated with the One Pot method previously described, however, is the inactivation of the nucleotide substrate in each cycle so that it will not interfere in subsequent cycles of the synthesis. Nucleotide substrate inactivation is necessary if a different base is to be added in the next cycle. However, if the same base is to be added in the next cycle, then it would be desirable to be able to skip the inactivation step and utilize the unincorporated nucleotide substrate in the next cycle, and perhaps several more ensuing cycles. No previous method exists, however, in which nucleotide substrate is reused, with or without intermediate purification. It is an object of the present invention to provide such a method.
The challenge presented by this objective is evident when it is considered that the same blocking group exists, in substantially similar chemical environments, on the 3'-end of the extended primer and on the 3'-end of unincorporated nucleotides, and that removal of the blocking group from the extended primer must be accomplished selectively, i.e., without substantial removal of the blocking group from unincorporated nucleotide substrate. Nevertheless, a successful approach has been devised which permits the selective removal of the blocking group from the primer-blocked nucleotide product, without removing the blocking group from and thus inactivating the nucleotide substrate. As a result, an oligonucleotide synthesis can be performed with less nucleotide substrate reagent consumption, and therefore, at a lower cost.